Sep 27, 2017
RNA extraction from Synechocystis sp. PCC 6803 with Trizol reagent
- 1Department of Chemistry - Microbial Chemistry, Ångström and Science for Life Laboratory, Uppsala University, Sweden
- CyanoWorld
Protocol Citation: Dennis DD Dienst 2017. RNA extraction from Synechocystis sp. PCC 6803 with Trizol reagent. protocols.io https://dx.doi.org/10.17504/protocols.io.j3scqne
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 27, 2017
Last Modified: March 15, 2018
Protocol Integer ID: 8018
Label all required tubes and store on ice (or freezer, if not needed immediately)
Pre-cool all required centrifuges
Fill sterile 50 mL tube (Falcon) w/ ice and store in ice bath
Pour 20-25 mL cell culture (OD750 < 1.0) to ice-filled tube (up to ~45 mL mark)Note: avoid long transport of cell culture before harvest
spin down at 4000 - 5000 g and 4 ° C for 5 min
discard supernatant (w/ ice) into big beakerNote: depending on strain/mutant some cells will get lost at this step
resuspend cell pellet in residual water (~ 1 mL)
transfer suspension into 2 mL (safe lock!) tubes (work on ice!)
spin down at ~13.000 g and 4° C for 15 sec
discard supernatant w/ pipetNote: try to remove supernatant 'as quantitatively as possible'
resuspend pellet in 1 mL Trizol reagent
store at -20 °C or (better) -80 °C
incubate frozen samples at 65° C for 15 min under constant agitationNote: if no shaking thermoblock is available, vortex once per minute
add 200 µL (ice cold) chloroform-isoamylalcohol (24:1) per 1 mL Trizol and vortex for 30 sec
spin down at ~11.000 g and 4° C for 10 min
transfer upper, aqueous phase to fresh 1.5 mL tube
add 1 Vol. (ice cold) phenol-chloroform-isoamylalcohol (25:24:1)Note: for RNA preparation phenol solution/mixtures should not be Tris-buffered
transfer upper, aqueous phase to fresh 1.5 mL tube
add 1 Vol. isopropanol (2-propanol), 10 µL 3 M Na-Acetat (pH 5.2) and 1 µL glycogen (RNA grade, Thermo)
incubate o/n at -20 for precipitation
spin down at ~13.000 g and 4° C for 30 min
remove supernatant and wash pellet w/ 70% EtOH (ice cold)
spin down at ~13.000 g and 4° C for 10 min
repeat steps 24 and 25
discard supernatant and air-dry RNA pellet for ~10 min
resuspend RNA Pellet in ~30 µL ultra-pure water and store at -20 or (better) -80°C